Download -Chain Gene in Epididymis α Expression of the C4b

Novel Androgen-Dependent Promoters Direct
Expression of the C4b-Binding Protein α
-Chain Gene in Epididymis
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of June 16, 2017.
Mayumi I. Nonaka, Guixian Wang, Takao Mori, Hidechika
Okada and Masaru Nonaka
J Immunol 2001; 166:4570-4577; ;
doi: 10.4049/jimmunol.166.7.4570
http://www.jimmunol.org/content/166/7/4570
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References
Novel Androgen-Dependent Promoters Direct Expression of
the C4b-Binding Protein ␣-Chain Gene in Epididymis
Mayumi I. Nonaka,*† Guixian Wang,† Takao Mori,* Hidechika Okada,† and Masaru Nonaka1*
T
he regulation of complement is essential to avoid its excessive activation and to protect self cells against complement attack. Many secretory and membrane-associated
regulators mediating such activity have been identified in plasma
and on the surfaces of blood cells and various epithelial cells. In
the reproductive organs the distribution of membrane-associated
complement regulators, such as decay-accelerating factor (DAF),2
membrane cofactor protein (MCP), and CD59, has been well characterized in humans (1– 6) and in guinea pigs (7, 8). These proteins
have been detected mainly in the female reproductive organs, such
as the uterus, oviduct, ovary, and placenta, although the expression
patterns of these proteins are distinct. All these proteins have also
been localized on spermatozoa. It has been suggested that these
regulators play a pivotal role in the protection of tissues and spermatozoa from complement attack caused by sperm Ag or invasive
micro-organisms in the female reproductive tract, where complement-hemolytic activity has been detected (1). Furthermore, a recent gene-targeting study indicated that Crry, another murine
membrane-associated complement inhibitor, plays a critical role in
protecting embryos from complement attack during pregnancy (9).
Recently, a sperm protein involved in sperm-egg interaction was
isolated from mice and guinea pigs, referred to as sp56 and AM67,
respectively (10, 11). This protein shows significant structural sim*Department of Biological Sciences, University of Tokyo, Tokyo, Japan; and †Department of Molecular Biology, Nagoya City University School of Medicine, Nagoya,
Japan
Received for publication October 16, 2000. Accepted for publication January
29, 2001.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
Address correspondence and reprint requests to Dr. Masaru Nonaka, Department of
Biological Sciences, University of Tokyo, Hongo 7-3-1, Tokyo 113-0033, Japan.
E-mail address: [email protected]
2
Abbreviations used in this paper: DAF, decay-accelerating factor; MCP, membrane
cofactor protein; C4BP, C4b-binding protein; C4BP␣, ␣-chain of C4BP; SCR, short
consensus repeat; SAP, serum amyloid protein; RACE, rapid amplification of cDNA
ends; UT, untranslated; ARE, androgen response element; HNF-1, hepatocyte NF-1.
Copyright © 2001 by The American Association of Immunologists
ilarity to the ␣-chain of C4b binding protein (C4BP␣), one of the
fluid phase complement regulators. This finding instigated our investigation of C4BP␣ in the reproductive organs.
Human C4b-binding protein (C4BP) is a large, hetero-oligomeric plasma glycoprotein (⬃550 kDa) and is recognized as an
acute phase protein. The major form is composed of seven
␣-chains (70 kDa) and one ␤-chain (45 kDa), which are disulfidelinked at their C-terminal regions. Each ␣-chain is composed of
eight short consensus repeat (SCR) domains, followed by the Cterminal region. The ␤-chain is composed of three SCR domains,
followed by the C-terminal region. C4BP binds to complement
component C4b through each ␣-chain and regulates complement
activation by decay acceleration of the C3 convertase of the classical pathway (C4b2a) and/or by acting as a cofactor for the cleavage of C4b by factor I (12–14). The ␤-chain contains a binding site
for the anticoagulant vitamin K-dependent protein S, which suggests that C4BP also acts as a regulator in the protein C coagulant
pathway (15, 16). Furthermore, it has been reported that the
␣-chain of C4BP interacts with serum amyloid protein (SAP) in
plasma (17), and that the binding of SAP to C4BP decreases the
complement regulatory activity of C4BP (18).
In this paper we describe the significant expression of C4BP␣ in
both guinea pig and murine epididymis and demonstrate the tissuespecific transcription of the guinea pig C4BP␣ gene from alternate
promoters. We describe here a novel interaction between complement regulators and the reproductive organs.
Materials and Methods
Molecular cloning of guinea pig C4BP␣
A cDNA library, constructed from poly(A)⫹ mRNA from the testis of an
approximately 7-wk-old guinea pig (19), was screened with a 0.2-kb
C4BP␣-like cDNA fragment homologous to the SCR2-SCR3 region of
human C4BP␣, which was unexpectedly isolated by RT-PCR during amplification of guinea pig DAF cDNA. The probe was labeled with
[␣-32P]dCTP using the Rediprime DNA labeling system (Amersham Pharmacia Japan, Tokyo, Japan), and hybridization was performed for 20 h in
1 M NaCl, 50 mM Tris-HCl (pH 8.0), 10 mM EDTA, 10⫻ Denhardt’s
solution, 1% salmon sperm DNA, and 0.1% SDS at 55°C. Thirteen positive
0022-1767/01/$02.00
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C4b-binding protein (C4BP) is a large plasma protein composed of seven ␣-chains and one ␤-chain and is involved in the fluid
phase regulation of the classical pathway of the complement system. Complement inhibitory activity is located in the ␣-chain, and
its mRNA has been detected only in liver to date. Here, we have isolated cDNA clones encoding the ␣-chain of guinea pig C4BP
(C4BP␣) and have demonstrated significant C4BP␣ mRNA expression in epididymis as well as liver. The level of C4BP␣ transcripts increased in the epididymis after birth, while it remained constant in the liver. C4BP␣ mRNA was also detected in the
normal murine epididymis at a significant level, but it decreased drastically after castration, suggesting that epididymal expression
of the C4BP␣ gene is regulated by androgen. Gene analysis of guinea pig C4BP␣ indicated that liver and epididymis C4BP␣
mRNA share the coding region and 3ⴕ-untranslated region, but are transcribed from independent promoters on a single-copy gene.
Two novel epididymis-specific promoters were identified in the region corresponding to the first intron of liver transcripts. The
binding motif for hepatocyte NF-1 occurs in the promoter used for transcription of liver C4BP␣, whereas androgen-responsive
elements occur in both promoters used in the epididymis. These findings present a novel link between complement regulators and
reproduction. Furthermore, variation in the 5ⴕ-untranslated regions, arising from alternative splicing of the newly identified exons,
is demonstrable in the guinea pig C4BP␣ transcripts. The Journal of Immunology, 2001, 166: 4570 – 4577.
The Journal of Immunology
plaques were isolated in the screening of 5 ⫻ 105 recombinant plaques.
These were plaque-purified and converted into plasmid clones by the inherent excision process according to the manufacturer’s directions. The
nucleotide sequences were determined using the chain termination method,
with an ALF DNA sequencer (Pharmacia LKB Biotechnology, Uppsala,
Sweden) or an ABI 370 DNA sequencer (Applied Biosystems, Foster
City, CA).
Guinea pig liver and epididymal C4BP␣ cDNA were isolated by 5⬘- and
3⬘- rapid amplification of cDNA ends (RACE), using the adapter primer
5⬘-GACTCGAGTCGACATCG-3⬘, a dT17-adapter primer, and terminal
transferase (Roche Japan, Tokyo, Japan). Other oligonucleotide primers
used were as follows: GBP-2 (5⬘-TGCCTGTCACTGTCACA-3⬘) and
GBP-4 (5⬘-TCTCCTGGATTTCTGCA-3⬘) for 5⬘-RACE, and GBP-1 (5⬘AAGTCATCTGTCGCCAG-3⬘) and GBP-3 (5⬘-CAAACAGTTGTGT
GGA-3⬘) for 3⬘-RACE. GBP-2 and GBP-1 were used for the RT of RNA.
Products amplified by 5⬘-RACE were gel-purified and subcloned into the
pCR2.1TOPO vector (Invitrogen, San Diego, CA). Products amplified by
3⬘-RACE, in both the liver and the epididymis, were detected as a single
band by agarose gel electrophoresis, and were directly sequenced after
pretreatment with exonuclease I and shrimp alkaline phosphatase (Amersham Pharmacia Japan).
Northern blotting analysis
RT-PCR amplification
The following oligonucleotides were synthesized: A1–1, 5⬘-CAAGGC
CCTAGGCACAC-3⬘; A3–5, 5⬘-AAAAGAGCGAGAGGTAT-3⬘; and
A5–1, 5⬘-GCTACTAGTCCACTTCA-3⬘, which bind to a site in the 5⬘untranslated (UT) region of the U1, U3, and U5 types, respectively (sense
primer). GBPA12 (5⬘-CTACAGAAGACCTCATA-3⬘) was designed to
bind a site in the common SCR1 region (antisense primer; shown by dotted
underlining in Figs. 2 and 4b). Total RNA from the liver, epididymis, and
testis was reverse transcribed, and cDNA fragments were amplified by
PCR, with denaturation at 95°C for 3 min, followed by 30 cycles of 95°C
for 0.5 min, 42°C for 0.5 min, and 72°C for 1 min and a final extension at
72°C for 5 min.
Isolation of genomic clones encoding the 5⬘-UT region of
guinea pig C4BP␣
A guinea pig genomic library constructed in the EMBL3 vector was purchased from Clontech (Palo Alto, CA). Fragments including the 5⬘-UT
regions of the isolated clones of guinea pig C4BP␣ were used as probes to
screen 1 ⫻ 106 plaques. Four clones were plaque-purified, and the phage
DNA was isolated using standard methods. The inserts were digested with
the appropriate restriction enzymes, subcloned into pGEM3Zf⫹ or pBluescript SKII vectors, and sequenced as described above.
RNase protection assay
Five or 10 ␮g of total RNA were hybridized with [␣-32P]CTP-labeled RNA
probes using the RNase protection assay kit RPAII (Ambion, Austin, TX).
Probes were labeled using RiboProbe In Vitro Transcription Systems (Promega, Madison, WI). PCR-amplified 382-, 417-, and 455-bp fragments in
the U5, U3, and U1 regions, respectively, were cloned into pGEM3Zf⫹,
linearized, and used to synthesize probes. However, the U3 probe did not
work well, detecting many nonspecific bands.
Results
Molecular cloning of guinea pig C4BP␣
Thirteen positive clones were isolated when a guinea pig testis
cDNA library was screened with a 0.2-kb fragment of guinea pig
C4BP␣, previously isolated by RT-PCR. These clones were divided into two groups according to the nucleotide sequences of
their coding regions. Four of these clones encoded seven SCRs
followed by the C-terminal region and were identified as the previously isolated sperm protein, AM67 (11). The other nine clones
encoded a new protein, composed of eight SCRs followed by a
C-terminal region, similar to C4BP␣ of other animals (except for
mice) (21–25), and the protein-coding region showed 57 and 73%
identity to that of human C4BP␣ at the amino acid and nucleotide
levels, respectively, suggesting that these clones encode a guinea
pig counterpart of human C4BP␣. However, guinea pig C4BP␣
cDNA included three transcript types with different 5⬘-UT regions,
termed U1, U3/U2, and U3 types (Fig. 1). The U3/U2 type 5⬘-UT
region occurs when the U2 sequence is inserted into the U3 type,
suggesting that guinea pig C4BP␣ mRNAs are transcribed from
two promoters. The schematic models of the isolated clones and
the percent identity between guinea pig C4BP␣ and AM67 in each
domain are shown in Fig. 1. Guinea pig C4BP␣ shows 55% overall
identity with AM67. It is noteworthy that the SCR2 and SCR3
regions of both show strong identity, since SCR2 and SCR3 are
important domains for binding to C4b (26). However, it is unclear
whether AM67 interacts with C4b.
The nucleotide and deduced amino acid sequences of the guinea
pig C4BP␣ cDNA are shown in Fig. 2. The amino acid identity
scores of each SCR between guinea pig C4BP␣ and that of other
animals, and between AM67 and sp56, the mouse homologue of
AM67, are summarized in Table I. In C4BP␣, overall identity
scores are relatively low, and the identity scores of each SCR vary
widely. On the other hand, sperm protein AM67/sp56 is generally
more conserved than C4BP␣, and the identity scores of each SCR
FIGURE 1. Schematic model of the cDNA clones encoding guinea pig C4BP␣ and AM67 isolated from a testis cDNA library. All cDNA clones
encoding C4BP␣ share an identical coding region, including signal peptides (SP), eight SCRs followed by the C-terminal (CT) region, and the 3⬘-UT region
(䡺). However, they contained three 5⬘-UT regions, termed U1, U3/U2, and U3. The U3 type lacks the U2 region in contrast to the U3/U2 type. The numbers
between C4BP␣ and AM67 represent the percentage of identity between each SCR at the amino acid (a.a., top) and nucleotide (nuc, bottom, in parentheses)
levels. The horizontal arrows indicate the probe sites used in Northern blotting analysis (see Fig. 3).
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Total RNA was isolated from various tissues of Hartley guinea pigs of
various ages (Japan SLC, Shizuoka, Japan) and from 10-wk-old BALB/c
mice (Japan Clea, Tokyo, Japan), using the guanidine thiocyanate/CsCl
method. Four 7-wk-old mice were castrated, and RNA was isolated on the
eighth day after castration. RNA was also isolated from three noncastrated
mice of the same age as controls. Approximately 5 ␮g of total RNA was
denatured with glyoxal and DMSO as described previously (20), separated
electrophoretically on 1% agarose gels, and transferred to Hybond-N nylon
membrane (Amersham Pharmacia Japan). Four cDNA fragments were
used as probes: a PCR-amplified 0.6-kb fragment of guinea pig C4BP␣,
corresponding to SCR5-SCR7; a 0.4-kb fragment of AM67, corresponding
to SCR5-SCR6; a 1.3-kb fragment of human GAPDH; and a PCR-amplified 1.0-kb fragment of murine C4BP␣, encompassing from SCR2 to the
C-terminal region. Labeling of the probes and hybridization were performed as described above.
4571
4572
EXPRESSION OF THE C4BP ␣-CHAIN GENE IN EPIDIDYMIS
are relatively constant, although SCR2 is its most highly conserved
domain. These results suggest a functional difference between
AM67/sp56 and C4BP␣.
Northern blotting analysis
To examine the tissue distribution of C4BP␣ and AM67, Northern
blotting analysis was performed using RNA from various tissues
of 14-wk-old guinea pigs and the 0.6- and 0.4-kb fragments of
C4BP␣ and AM67, respectively, as probes (shown as Probe A and
Probe B in Fig. 1). These probes did not cross-hybridize under the
experimental conditions described. As shown in Fig. 3A, significant expression of C4BP␣ mRNA was observed in the epididymis
as well as the liver, but not in the testis, whereas AM67 mRNA
was detected only in the testis.
Further analysis, using RNA taken from tissues at various developmental stages, indicated that epididymal C4BP␣ transcripts
increase significantly after birth, in concert with the testicular androgen levels (27), whereas liver C4BP␣ transcripts are expressed
at constant levels at all ages tested. This implies that the expression
of epididymal C4BP␣ is affected by androgen, and the expression
Table I. Amino acid identities (%) in each SCR between guinea pig
C4BP␣ and its counterparts in other animals (21–25) and between
AM67 and sp56
Guinea Pig C4BP␣
AM67
Human
Mouse
Rat
Rabbit
Bovine
sp56
SCR1
SCR2
SCR3
SCR4
SCR5
SCR6
SCR7
SCR8
CT
59
64
69
65
54
34
47
66
60
45
57
64
40
⫺a
⫺
44
64
42
53
66
73
53
55
33
51
66
56
55
68
75
66
45
36
42
64
64
50
57
71
63
45
35
43
63
56
69
79
65
63
57
68
⫺
63
50
Total
57
51
56
57
53
66
a
Mouse C4BP␣ lacks the domains corresponding to SCR5 and SCR6.
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FIGURE 2. a, Nucleotide and deduced amino acid sequences of the U3/U2 type of guinea pig C4BP␣ cDNA. N-terminal residue of guinea pig C4BP␣
shown as ⫹1 was estimated by alignment with other species C4BP␣. The potential signal peptide sequence is written in italics. Although Met(⫺55) is the
first ATG codon from the 5⬘end, Met(⫺12) is more likely to be the translation start site, since the sequence written in italics contains many hydrophilic
amino acids. Five potential N-glycosylation sites were found in SCR1, SCR3, SCR4, and SCR8, as shown by arrowheads. The primers used for 5⬘- and
3⬘-RACE are shown by underlining with directions, and the primers used for RT-PCR (Fig. 5) are shown by dotted underlining with directions. The
boundaries of each domain, indicated by arrows, were determined by evaluation of the exon/intron boundaries. b, Nucleotide sequence of the 5⬘-UT region
of the U1 type C4BP␣ cDNA. The primers used for RT-PCR (Fig. 5) are shown by dotted underlining with direction. The sequences shown have been
entered in the EMBL/GenBank/DDBJ database under accession numbers AB049465 and AB049466.
The Journal of Immunology
4573
of liver C4BP␣ is constitutive. In testis, the expression of C4BP␣
was very low at all ages (Fig. 3B), although extended autoradiographic exposure detected a faint band at 12 days of age (data not
shown). On the other hand, AM67 transcripts were detected from
9 wk of age, with no increase thereafter (Fig. 3B), suggesting that
AM67 mRNA is expressed in round spermatids, as has been reported for sp56 (10). The distinct tissue distributions of C4BP and
AM67 suggest a functional difference between them.
Isolation of epididymal and liver C4BP␣ cDNA by 5⬘- and
3⬘-RACE
Because the transcript size of epididymal C4BP␣ is smaller than
that of liver C4BP␣, as shown in Fig. 3, epididymal and liver
C4BP␣ cDNAs were isolated by 5⬘- and 3⬘-RACE. Sequence analysis of the isolated cDNAs indicated that epididymal C4BP␣
cDNA is identical with testis C4BP␣ cDNA, including the 5⬘-UT
sequences. However, although liver C4BP␣ cDNA was identical
with the epididymal and testis C4BP␣ cDNAs in the protein coding and 3⬘-UT regions, they differed in the 5⬘-UT region. The
5⬘-UT region of liver C4BP␣ transcripts included one of two sequences, termed U5 and U5/U4. The sequence U5/U4 occurred
when the U4 sequence was inserted at the 3⬘-end of the U5 sequence, suggesting that these two mRNA species are transcribed
from a common promoter. The schematic model of the epididymal
and liver C4BP␣ and the sequence of the 5⬘UT region of the
U5/U4 type are shown in Fig. 4. The sequence U5 is highly homologous (⬎65%) to the 5⬘-UT region of C4BP␣ of other animals,
except for bovine C4BP␣ (21–25), indicating that the U5 type
transcript corresponds to C4BP␣ mRNA of these animals. The
5⬘-UT sequence of bovine C4BP␣ showed no significant homology with any of the 5⬘-UT sequences of guinea pig C4BP␣ mRNA.
To confirm the tissue specificity of these mRNA species and to
examine which mRNA species are dominant in each tissue, RTPCR was performed using oligonucleotides designed to bind sites
in the U1, U3, and U5 regions as sense primers and to a site in the
SCR1 region as an antisense primer (underlined in Figs. 2 and 4b).
As shown in Fig. 5, the U1 type transcript was detected in the
epididymis and the testis, but not in the liver. A band corresponding to the U1/U2 type transcript, which was not isolated during
cDNA cloning, was also detected, although the level of expression
seemed very low. Similarly, the U3/U2 and U3 type transcripts
were detected at significant levels in the epididymis and only
FIGURE 4. a, Schematic model of the cDNA clones isolated from epididymis and liver by 5⬘- and 3⬘-RACE. The nucleotide sequences of the epididymis
C4BP␣ cDNAs were identical with those of testis C4BP␣ cDNAs, including all three variants in the 5⬘-UT regions. However, liver C4BP␣ cDNA contained
the different 5⬘-UT sequences, termed U5 and U4. Two classes were detected in the liver, determined by the presence or the absence of the U4 region. b,
The sequence of the 5⬘-UT region of liver C4BP␣ cDNA. A dotted underline indicates the position of the primer used for RT-PCR (see Fig. 5). The entire
sequence of the U5/U4 type has been entered in the EMBL/GenBank/DDBJ database under accession number AB049467.
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FIGURE 3. Northern blotting analysis of guinea pig C4BP␣ and AM67. a, Distribution in various tissues. The positions used in probes are indicated
in Fig. 1. Significant expression of C4BP␣ mRNA was observed in epididymis as well as in liver, but not in testis, while AM67 mRNA was detected only
in testis. b, Expression at the various developmental stages. The level of C4BP␣ mRNA significantly increased in the epididymis after birth, while it
remained constant in the liver. AM67 was detected after 9 wk of age, with no increase thereafter, suggesting that AM67 mRNA is expressed in round
spermatids. 12day, 12 days old; 4w, 4 wk old; 9w, 9 wk old; 14w, 14 wk old; 12 m, 12 mo old.
4574
faintly in the testis, but not in the liver. The levels of the U3/U2
and U3 type transcripts were almost equal. Conversely, the U5 and
U5/U4 type transcripts were detected preferentially in the liver,
although faint bands were also observed in the epididymis. Interestingly, the U5/U4 type transcript, but not the U5 type, was dominant in the guinea pig liver, while the U5 type transcript corresponds to C4BP␣ mRNA of other animals.
Analysis of the guinea pig C4BP␣ gene
To clarify the organization of the multiple 5⬘-UT regions of guinea
pig C4BP␣ mRNA, four genomic clones encoding these areas
were isolated and analyzed. As shown in Fig. 6, the restriction
enzyme maps of these clones were consistent with each other,
indicating that the guinea pig C4BP␣ gene is a single copy gene.
The exon/intron boundaries for the exons encoding a part of the
5⬘-UT and the signal peptide region (5⬘UT/SP), SCR1, and SCR2a,
FIGURE 6. Partial structure of the
guinea pig C4BP␣ gene. Four overlapping
clones, ␭1–␭4, were isolated from a
guinea pig genomic library, and ␭2 was
further analyzed by sequencing. The sequenced region is shown by a horizontal
arrow (EMBL/GenBank/DDBJ accession
number AB049468). The nucleotide sequences at the exon/intron boundaries are
shown below. Exons encoding U5 and
5⬘UT/SP, which show sequence homology to human and mouse C4BP␣, corresponded to the first and second exons of
the human and mouse C4BP␣ gene, respectively. Exons encoding the U4, U3,
U1, and U2 regions were newly identified
in the guinea pig C4BP␣ gene. Three putative promoters were identified in the 5⬘flanking regions of U5, U3, and U1.
were found in the same positions as the second, third, and fourth
exons, respectively, of the human and mouse C4BP␣ genes. The
exon encoding U5 corresponds to the first exons of the human and
mouse C4BP␣ genes and shows ⬎60% homology with them over
the 500 or more base pairs analyzed to date (28, 29). The exons
encoding U3 and U1 were found separately between the exons
encoding U5 and 5⬘UT/SP, suggesting that three independent promoters exist in the guinea pig C4BP␣ gene, and that tissue-specific
expression of C4BP␣ is regulated by differential use of these promoters. The U4 sequence was encoded by one exon, located close
to the 5⬘- end of the U3 region, and alternate splicing of this exon
generates two C4BP␣ mRNA species in the liver. The U2 sequence was found spliced to the 5⬘-end of the 5⬘UT/SP exon, indicating that use of alternative splice acceptor sites produces two
species in both epididymal C4BP␣ mRNAs transcribed from two
independent promoters.
Characterization of the promoter regions
To determine the transcription-initiation sites and to confirm the
tissue specificity of these promoters, an RNase protection assay
was performed using probes containing the U5 and U1 regions
together with their 5⬘-flanking regions. As shown in Fig. 7a, the
U5 probe was protected by transcripts expressed in the liver, and
the U1 probe was protected by transcripts expressed in the epididymis. In addition, both promoters contained multiple transcriptioninitiation sites.
Fig. 7b shows the nucleotide sequences of three promoter regions. The approximate positions of the transcription-initiation
sites described above are shown by arrows. In the U5 promoter
region, a hepatocyte NF-1 (HNF-1) response element, GTTAAT
NATTAAC, was found in the same position as those in the human,
mouse, and rat C4BP␣ genes (28 –30), explaining the preferential
transcription from this promoter in the liver (31). A TATA box
was found at the same position as that in the mouse C4BP␣ gene
(29). On the other hand, an androgen response element (ARE)
consensus sequence (32), GG(AT)ACANNNTGTTCT, was found
with three mismatches in each of the 5⬘-flanking regions of U1 and
U3, explaining the androgen-dependent transcription from these
promoters, in the epididymis. A TATA box was also found in both
these regions.
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FIGURE 5. RT-PCR analysis of guinea pig C4BP␣ mRNA expressed
in liver (Li), epididymis (Ep), and testis (Te). Total RNAs were reverse
transcribed and subjected to PCR amplification with oligonucleotide primers specific for the U1 (a), U3 (b), and U5 (c) types (shown by dotted
underlining in Figs. 2 and 4b). Amplified products were analyzed on 2%
agarose gels with the size marker (M). The U1 type is expressed in epididymis and testis. The U3/U2 and U3 types are preferentially expressed in
epididymis, while the U5/U4 and U5 types are preferentially expressed in
liver. The U1/U2 type that was not isolated by 5⬘-RACE was detected by
RT-PCR in epididymis and testis.
EXPRESSION OF THE C4BP ␣-CHAIN GENE IN EPIDIDYMIS
The Journal of Immunology
4575
Androgen-dependent expression of murine C4BP␣ in the
epididymis
To investigate whether the significant expression of C4BP␣ in the
epididymis is specific to guinea pigs, we examined the tissue distribution of murine C4BP␣ mRNA. As shown in Fig. 8A, normal
adult mice express a significant level of C4BP␣ mRNA in the
epididymis as well as in the liver in a pattern similar to that in
guinea pigs. To confirm that the expression of epididymal C4BP␣
mRNA depends on androgen, we analyzed the C4BP␣ transcript
levels in castrated mice. As shown in Fig. 8B, the level of epididymal C4BP␣ mRNA was significantly reduced in castrated mice
compared with that in noncastrated mice, while transcript levels in
the liver showed no significant difference, suggesting that expression of epididymal C4BP␣ mRNA is androgen dependent.
Discussion
C4BP␣ is the major component of C4BP participating in the
fluid phase regulation of the complement system, and its mRNA
had been detected only in liver. In this report we describe
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FIGURE 7. Analysis of the promoter regions.
a, RNase protection assay. The transcription initiation sites of the U5 and U1 types were determined, using as probes the 382- and 455-bp fragments containing U5 and U1 sequences,
respectively. The U5 and U1 promoters are tissue specific, and both promoters include multiple transcription initiation sites (indicated by
arrows). b, Nucleotide sequences of three promoter regions. The positions of the transcriptioninitiation sites determined above are shown by
arrows. The 5⬘-ends of the cDNAs encoding the
U3/U2 and U3 types obtained by 5⬘-RACE are
dotted. The putative TATA boxes are underlined. The binding motifs for HNF-1 (GTTAAT
NATTAAC) and the ARE (GGWACANNNT
GTTCT) are shown by double underlining.
Exon/intron recognition signals, AG and GT, are
shown in lowercase.
significant androgen-dependent expression of guinea pig and
murine C4BP␣ mRNA in the epididymis, one of the male reproductive organs. Analysis of the cDNA sequences and the gene
structure of guinea pig C4BP␣ indicates that epididymal and liver
C4BP␣ mRNA are identical in their coding regions, 3⬘-UT regions, and parts of their 5⬘-UT regions. However, they are transcribed in a tissue-specific manner from independent promoters on
a single-copy gene.
Guinea pig C4BP␣ cDNA clones were first isolated from a testis
cDNA library. However, further investigation using Northern blotting (Fig. 3), RT-PCR (Fig. 5), and RNase protection analyses (Fig.
7a) indicated that C4BP␣ mRNA is poorly expressed in this tissue. As
we could not eliminate the possibility of contamination of the caput
region of the epididymis in testis sections during tissue preparation,
the precise distribution should be determined more definitively using
other methods, such as immunohistochemistry. Although mRNA of
AM67, which is structurally related to C4BP␣, is expressed in testis,
the difference in tissue distribution between C4BP␣ and AM67 is
clearly indicated (Fig. 3).
4576
Guinea pig C4BP has been isolated previously from acrosomeintact spermatozoa (33), as well as from plasma (34). When guinea
pig fertilin (as called PH-30), which is a potential sperm-egg membrane fusion protein, was isolated from cauda epididymal spermatozoa using an mAb column against fertilin, two additional proteins coeluted. One was identified as a new member of the pentaxin
protein family, termed apexin. The other was shown to be homologous to human C4BP using the SDS-PAGE, Western blotting,
and amino acid sequence analyses. However, since its mobility
was slightly different from that of serum C4BP, it was termed
sp-C4BP. The two partial amino acid sequences of sp-C4BP described in that report, EGGYLSALSYVYECDDGYTLVGQN and
NPGDLPHGTIEVK, are completely identical with regions of the
amino acid sequence of guinea pig C4BP␣ deduced from the
cDNA clones isolated here (Fig. 2), indicating that sp-C4BP is
definitely guinea pig C4BP. The slight difference observed in their
motilities may be the result of post-translational modification, such
as glycosylation. Furthermore, since C4BP␣ mRNA was detected
at negligible levels in adult testes, sp-C4BP is likely to originate in
the epididymis and attached to spermatozoa via apexin or fertilin,
or directly.
Although it is unclear whether the association of C4BP, apexin,
and fertilin is physiologically relevant (33), it is tempting to speculate that epididymal C4BP may interact with apexin in a way
comparable to the complex formed by serum C4BP with SAP,
another member of the pentaxin family (17, 18). However, because
apexin is an intracellular protein, located in the acrosomal region
(33), it is unlikely that epididymal C4BP binds to apexin during the
passage of spermatozoa through the epididymis. The binding of
epididymal C4BP to apexin may be an artifact arising during protein isolation. Otherwise, C4BP may interact with fertilin, which is
expressed on the surface of the spermatozoa and proteolytically
processed during sperm maturation in testis and epididymis (35).
Analysis of the guinea pig C4BP␣ gene identified three promoters. One is used in liver and shows significant sequence homology
to the promoters already reported for the human, mouse, and rat
C4BP␣ genes. All these promoters contain an HNF-1 consensus
sequence (28 –30) that is essential for the hepatic activity of the
promoter of the C4BP␣ gene (31). The other two promoters are
newly identified in the C4BP␣ gene and are transcriptionally active in the epididymis. Neither of the promoters active in the epididymis contains consensus sequence for HNF-1, but they do contain the consensus sequence for an ARE. The promoter used in
liver contains no consensus sequence for ARE. These findings support the tissue-specific expression of C4BP␣ mRNA and explain
why epididymal C4BP␣ mRNA is transcribed in an androgendependent manner.
The guinea pig C4BP␣ gene is unusual in that it generates several mRNA species by alternate splicing of the newly identified
5⬘-UT exons, which occur between the exons corresponding to the
first and second exons of the human and mouse C4BP␣ genes. As
a result of this alternate splicing of the additional 5⬘-UT exon, liver
C4BP␣ transcripts possess at least two possible 5⬘-UT sequences.
On the other hand, utilization of alternate splice acceptor sites in
the exon encoding the U2 and 5⬘-UT/SP regions in combination
with the use of alternate promoters produces at least four epididymal C4BP␣ transcripts with different 5⬘-UT regions, as detected
by RT-PCR (Fig. 5). Such heterogeneity in the 5⬘-UT region has
been found in many proteins. In the expression of acetyl-coenzyme
A carboxylase, it is considered to be involved in the regulation of
translation (36). However, the reason why the 5⬘-UT region of
C4BP␣ mRNA is heterogeneous in guinea pigs, but not in other
animals, remains unclear.
Significant androgen-dependent expression of C4BP␣ in the epididymis is observed not only in guinea pigs but also in mice,
suggesting a novel role for C4BP in reproduction. No other reproductive organs, such as seminal vesicle, uterus, or ovary, express
C4BP␣ mRNA at detectable levels in either guinea pigs (Fig. 3A)
or mice (Fig. 8A and our unpublished observations). Guinea pig
epididymal C4BP exists as a high molecular mass oligomeric protein (540 –590 kDa), similar to serum C4BP (33, 34), suggesting
that epididymal C4BP may possess a complement inhibitory activity. Clusterin (also known as SP-40), which is one of the major
secreted proteins in the epididymis, has been thought to function as
an inhibitor of the membrane attack complex in the complement
system (37). However, clusterin is ubiquitously expressed (38),
and recent findings indicate that under physiological conditions
clusterin is unlikely to be an important complement regulator (39).
Since clusterin possesses multiple functions besides complement
inhibition, such as its roles in the regulation of apoptosis and in
lipid transport (38, 39), it may play a noncomplement role in epididymis. Therefore, C4BP seems to be the first complement regulator to be identified as an epididymal protein, and the first fluid
phase complement inhibitor synthesized in the reproductive organs
to be identified.
On the other hand, the epididymis is protected from the immune
system by the blood-epididymis barrier, similar to the blood-testis
barrier under normal conditions, and no complement has been detected in this organ (1, 37). Therefore, if epididymal C4BP functions as a complement regulator, it may bind to spermatozoa and
play a role in the protection of spermatozoa in the female reproductive tract, where complement activity has been detected (1).
However, since spermatozoa express many membrane-associated
complement inhibitors, such as DAF, MCP, and CD59, on the
entire surface and/or on the inner membrane (4 – 6, 8), further association of C4BP is unlikely to be essential for protection of the
spermatozoa. C4BP may be involved in sperm maturation.
In this context other roles in reproduction have been speculated
for some of the complement regulatory proteins, over and above
their roles in complement regulation. For example, it has been
suggested that human MCP, expressed on spermatozoa, is involved in sperm-egg recognition (40, 41). Negligible expression of
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FIGURE 8. Northern blotting analysis of mouse C4BP␣. a, Distribution
in various tissues. Normal 10-wk-old mice express a significant level of
C4BP␣ mRNA in epididymis as well as in liver, similar to guinea pigs. The
upper faint band corresponds to the mRNA species containing additional 1
kb in the 3⬘-UT region (data not shown). b, Expression in the castrated
mice. Four 7-wk-old male mice were castrated, and RNAs were isolated 10
days after castration. The level of C4BP␣ mRNA in the castrated mice was
very low compared with that in normal mice in the epididymis (Ep), while
no significant difference was observed in the liver (Li), suggesting that
expression of C4BP␣ mRNA in the epididymis is androgen dependent.
EXPRESSION OF THE C4BP ␣-CHAIN GENE IN EPIDIDYMIS
The Journal of Immunology
rodent MCP in any cells except spermatozoa also suggests a specific role for this protein in reproduction (19, 42, 43). In addition,
guinea pig DAF is significantly expressed on the epithelial cells of
seminal vesicle, where no complement has been detected (7, 8).
This distribution is suggestive of a novel function for DAF. Furthermore, human C4BP␤, but not C4BP␣, was recently detected in
the regressing corpus luteum and corpus albicans of the adult human ovary (44). As C4BP␤ contains no complement inhibitory
activity, ovarian C4BP␤ is obviously involved in a system distinct
from the complement system. These proteins, C4BP␣, C4BP␤,
MCP, and DAF, as well as sperm protein AM67/sp56 are all composed of SCR domains. The SCR proteins might have developed
in close contact with the reproductive system. Further investigation
of the roles of these proteins in reproduction would define the
diversified functions of the SCR proteins recognized as complement regulators.
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